PROJECT SUMMARY Polyps are hyperproliferative lesions of the colorectal epithelium that have the potential to progress to carcino- mas. The classic normal-mucosa-polyp-carcinoma sequence has traditionally focused on the genomic changes involved in advanced stages of intestinal carcinogenesis and has not adequately addressed the transcriptomic events involved in the transformation of normal colorectal epithelium into benign hyperproliferations and then into high-risk polyps. Addressing this lack of knowledge is crucial to distinguish genomic events and pathways that promote progression of benign overgrowths from those lesions that harbor malignant potential, thus help- ing to improve our risk stratification of polyps. In addition, the relationships between the transcriptomic profiles of the different molecular subtypes of colorectal carcinomas and the transcriptomic profiles of polyps remain unexplored. The objective of this application (aimed at addressing Provocative Question 6) is to identify genes and pathways that are involved in the transition from benign overgrowths into malignant progression by com- paring the gene expression profiles of hyperplastic polyps with polyps with malignant features using next- generation sequencing coupled with integrative systems biology tools, and then to validate their biological role in carcinogenesis using patient-derived colorectal organoids. Our central hypothesis is that comparative gene expression analyses of at-risk normal mucosa, benign hyperplastic polyps, and polyps with higher levels for malignant progression will reveal pathways that are essential for promotion intestinal carcinogenesis being complementary to classical cancer driver genes. The rationale supporting this proposal is based on our: 1) In- tegrative analysis of gene expression data sets that have identified a subset of benign hyperplastic polyps that display a similar transcriptomic profile to normal mucosa; 2) In silico results distinguishing benign hyperplastic polyps without malignant potential from other polyp types based on the deregulation novel candidate genes; 3) Systems biology analyses that have identified specific molecular subtypes of CRC that are connected to well- known molecular carcinogenesis pathways. We propose to test our central hypothesis by investigating the fol- lowing three specific aims: 1) determine the transcriptomic profile of hyperplastic polyps and compare it with profiles from adjacent mucosa and polyps with higher malignant potential, 2) develop a genomic classification of premalignant colonic lesions according to colorectal cancer subtypes, and 3) validate the biologic role of candidate genes that are differentially expressed between hyperplastic polyps and high-risk lesions using pa- tient-derived colorectal organoids modified via CRISPR-Cas9 approaches. The proposed research is innova- tive in that it will use massive parallel RNA-sequencing in combination with systems biology tools to identify pathways related to colorectal hyperproliferation and to validate the role of those candidate genes using or- ganoids. The proposed research is significant in that it will facilitate discovery of novel biomarkers that distin- guish benign from premalignant polyps helping to refine the risk prediction for colorectal cancer development.